Protein phosphatase -1 and oocyte maturation: Regulation of germinal vesicle breakdown and chromatin condensation.

Abstract

The overall hypothesis of this dissertation is that inhibition of protein phosphatase-1 (PP1) is required for oocyte germinal vesicle breakdown (GVBD), but maintenance of PP1 activity is necessary for normal metaphase chromatin condensation. To address this hypothesis, we microinjected oocyte nuclei with neutralizing antibodies against PP1alpha and PP2A. Microinjection of PP1alpha antibodies increased oocyte GVBD compared to controls. If reduction of PP1alpha activity is important for GVBD, one would anticipate an endogenous means of reducing PP1alpha activity at this developmental stage. Phosphorylation of PP1alpha at Thr320 by cyclin dependent kinase-1 (CDK1) causes PP1alpha inactivation. Germinal vesicle-intact oocytes did not contain phosphorylated PP1alpha, however, in conjunction with GVBD oocyte PP1alpha became phosphorylated at Thr320 via CDK1 activity. Thus, inhibition of PP1alpha induces oocyte GVBD and PP1alpha activity during the event appears to be regulated via phosphorylation by CDK1. We then demonstrated OA inhibition of mouse oocyte PP1/PP2A caused aberrant chromatin condensation, probably as the result of histone-H3 at both ser10 and ser28 hyperphosphorylation. This may explain inability to progress past MI. PP1delta but not PP2A associated with oocyte condensed chromatin and recombinant PP1, but not PP2A dephosphorylated oocyte histone-H3. Furthermore, the PP1 regulatory protein, inhibitor-2 (Inh-2), was identified in oocytes at condensed chromatin during meiosis. Inhibition of oocyte glycogen synthase kinase-3 (GSK-3) appeared to regulate phosphorylation of Inh-2 and subsequent PP1 activity. Inhibition of GSK-3 also resulted in aberrant chromatin condensation, similar to that observed following OA treatment. These data indicate PP1delta regulates oocyte chromatin condensation and histone-H3 phosphorylation, and PP1 activity during these events may be regulated via Inh-2 and GSK-3. These are the first reports of distinct functional roles for PP1 isoforms and their endogenous regulation in mammalian oocytes. Together, these data may provide insight into meiotic signaling pathways, which may lead to therapies improving fertility and assisted reproductive technologies.